Arndt Asperger, Senior Applications Scientist, Bruker Daltonics and Meike Hamester, Vice President Label-Free Technologies, Bruker Daltonics, examine how challenges in mass spectrometry for high-throughput screening can be overcome using a label-free approach.
Mass spectrometry is inherently appealing for high-throughput screening (HTS), because it avoids certain drawbacks of label-based assays, but long-standing limitations of cost per sample, sample throughput and specificity have limited its application. These problems can be overcome by a label-free MS-based HTS approach combining the speed and proven robustness of matrix-assisted laser desorption ionization (MALDI) sampling with rapid separation of isobars and isomers by trapped ion mobility spectrometry (TIMS), and accurate-mass detection by high-resolution quadrupole time-of-flight mass spectrometry (QTOF MS). The method’s very high assay specificity enables the identification of high-quality leads at HTS-relevant speed and cost. Together with its unique capabilities for collisional cross-section (CCS)-enabled near-real time high-throughput experimentation (HTE) chemical synthesis monitoring, and the expanded chemical space provided by MALDI-based laser post-ionization, timsTOF technology stands to improve the efficiency of drug development in lead discovery and drug molecule design.
The rapid evolution of HTS to meet industry demand
HTS as a tool for drug discovery has evolved rapidly since the origin of the technique in the 1990s. HTS is widely used by pharmaceutical companies to identify compounds (or ‘hits’) that show pharmacological activity, which are used as starting points to optimise medicines during the drug discovery process.1 A particularly striking trend has been the growth in the number of compounds that can be tested per day, which is now routinely in the hundreds of thousands, mirroring the adoption of microliter-volume 384-well and then 1536-well plates,2 along with the introduction of automation technologies.
Figure 1: Separation principle of trapped ion mobility spectrometry: Ions are trapped in TIMS tunnel 1, are then transferred to and spatially focused in TIMS tunnel 2 (left), from where they are sequentially released according to their CCS by time-resolved reduction of the electric field strength (right).One of the other limiting factors for HTS – cost per well analysed – has also been addressed. Reducing assay volumes from about 200 µL to 1 µL has cut the quantities of reagents and consumables needed, while automation has replaced manual handling, so lowering running and personnel costs.
Figure 2: Quantitative MALDI-TIMS-MS analysis of glucose in the presence of the isomer fructose, demonstrating quantitation of isomers that would be indistinguishable by mass spectrometry alone.Due to the success of these approaches, the ability of HTS to deliver useful results is now increasingly limited by the quality of the hits obtained. The number of false-positive results is particularly problematic when screening large compound libraries, and can lead to wasted time and resources. Running secondary screens for confirmation is one approach, but this adds to the amount of effort involved. Scientists therefore need strategies that can provide high-quality hits without impacting either throughput or cost.
Overcoming challenges in HTS using mass spectrometry
One way to achieve this is the use of advanced read-out methods. By far the majority of HTS studies use fluorescence or luminescence methods for this purpose, which although sensitive, fast, and easily automated, have a number of drawbacks. For example, optimising the colorimetric labels is complex because they can interfere with the target biology and lead to false positives/negatives, while compounds containing chromophores in the relevant region of the spectrum cannot be included in the screen.
Figure 3: Quantitative analysis of a drug-like molecule (MW 543 Da), showing the elimination of interfering assay background and the consequent improvement in quantitation using TIMS in conjunction with MALDI-MS/MS. Sample courtesy of Dr Frank H. Büttner and colleagues, Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.Mass spectrometry is an alternative read-out technology that has great appeal for HTS, because it directly measures the mass-to-charge (m/z) ratio of analytes, enabling high selectivity and excellent sensitivity for virtually any system in vitro, without the need for labeling. Furthermore, MS provides access to a uniquely wide drug target space, which in part is not addressable by label-based HTS methods.3,4 As a result, setting up a screen is much faster, the risk of false positives and false negatives is greatly reduced, and confirmatory screens are rendered unnecessary. In addition, the MS read-out process inherently generates information about the target, providing a head start for any lead optimisation studies.
MALDI is a ‘soft’ ionization technique that provides high responses for the unfragmented molecular ion and can be applied to a broad variety of molecules ranging from small drug-like compounds up to larger peptides and intact proteins.5 Required sample volumes are typically in the sub-microliter range and, when partnered with TOF mass analysers, MALDI-based MS provides high sensitivity, mass accuracy and mass resolution, along with a broad dynamic range. Consequently, since the early 2000s MALDI-TOF MS has become widely used in the biomolecular field, principally for clinical microbiology and imaging mass spectrometry of tissue sections.6,7
However, despite this long list of features that makes MALDI-TOF MS suitable for drug screening, it was not until 2014 that the first study detailing its use in a high-throughput assay was published.8 This is largely because improvements in laser technology achieved over the last decade mean that lasers for MALDI applications can now operate at frequencies as high as 10 kHz, which allow run times to be reduced to well below 1s per sample. This has sparked interest in using MALDI-TOF MS for HTS and ultra-HTS applications in academia and industry,9 with developments including automation in a 1536-well format,10 biochemical assays for primary screening of ultra-large libraries comprising more than a million compounds,11 cell-based assays,12 phenotype screening,13 and binding assays.14
Trapped ion mobility spectrometry: Improving the specificity of MALDI-MS-based HTS
The ability of MALDI-enabled TOF and QTOF systems to discriminate between analytes and sample matrix background components based on their specificity is, like other MS techniques, dependent upon the mass resolution. Modern instruments offer routine resolutions of 50,000 with uncompromised sensitivity, but molecules with masses so close that they cannot be separated even with this resolution (isobars), and specifically analytes that have the same molecular formula (isomers), cannot be easily discriminated from each other by mass spectrometry alone. This imposes the need for upfront separation methods, but these can again cause conflict with the challenging analysis speeds required in HTS measurement campaigns.
Figure 4: MALDI-TIMS-MS(/MS) analysis of Pimozide, an antipsychotic drug: (left) mass spectrum providing accurate mass and isotopic pattern information; (center) trapped ion mobility spectrometry data (CCS); (right) MS/MS fragment ion spectrum. The mSigma value indicates the level of isotopic fidelity of a measured MS signal versus the theoretical isotope pattern of a target compound calculated from its molecular formula (lower is better).Recent timsTOF technology that couples TIMS to high-resolution QTOF mass spectrometers is now promising to remove this limitation.15 TIMS is an ion mobility spectrometry technique that separates ions in the gas phase according to their CCS – a measure of how likely they are to be deflected by collision with buffer gas molecules as they drift through an ion tube under the influence of an electric field.16 Because molecular ions of isobars (and even isomers) will have different CCS values, coupling TIMS to a QTOF system provides an orthogonal separation approach that addresses the specificity issue in HTS assays while keeping the analysis speed high.
In contrast to other ion mobility technologies, TIMS uses a buffer gas flow as the driving force in opposition to a reducing electric field, which causes the ions to be released in ‘packets’ into the mass analyser, depending on their CCS value (Figure 1).17 This lossless ion accumulation provides greater separation efficiency, resulting in a resolving power of up to R = 200, making it ideal for challenging analyses of isobars and isomers in complex samples. Use of timsTOF technology has, therefore, triggered a paradigm change in life science analytics, enabling new levels of depth and coverage, for example in TIMS enabled 4D omics and tissue imaging MS applications.
Maintaining the rapid analysis times needed for HTS
Unlike separation by chromatography, separation by TIMS is fast, taking place on a timescale of a few hundred milliseconds. As a result, the addition of TIMS to a MALDI-based MS system does not significantly increase the analysis time. In contrast to other hyphenated systems that would be needed to achieve this level of resolution, the timsTOF setup, when utilising fast MALDI sampling, can therefore be readily incorporated into HTS workflows. Examples of the specificity that can be achieved using this approach are shown in Figure 2 and Figure 3.
Keeping pace with synthetic chemistry HTE in support of drug molecule design
Another emerging application area of timsTOF technology in the pharmaceutical industry is synthetic chemistry HTE, which involves developing large libraries of newly designed molecules by running large numbers of chemical reactions in parallel.18 However, a bottleneck in this process is analysing and confirming the identity of reaction products in a sufficiently rapid timeframe. timsTOF technology in combination with fast MALDI sampling is capable of keeping pace with HTE chemical synthesis, and provides results in near-real-time, not just on the accurate mass of synthesised products, but on other physical properties such as isotopic pattern, CCS, and (if needed) MS/MS fragmentation data (Figure 4). This enhances the confidence level of product verification, and at the same time reduces turnaround time and chemical costs.19,20
Figure 5: Enhanced ionization of estradiol under laser induced post-ionization conditions, resulting in a gain of signal intensity of multiple orders of magnitude.A recently developed technique of laser induced post-ionization21further extends the chemical space accessible in HTE synthesis monitoring. The method involves firing a second laser into the evolving plume created by the initial MALDI pulse, resulting in a large gain in sensitivity for certain compounds that used to be out of scope of conventional MALDI.22 For example, estradiol, a sex-steroid hormone barely detectable in MALDI, yields an ion signal intensity that is orders of magnitude higher under post-ionization conditions (Figure 5).
The future of HTS
Using timsTOF technology as an HTS read-out method has the potential to significantly enhance the efficiency of the drug development process by increasing lead quality and providing access to a new drug target space in early drug discovery. The improved assay specificity and information content, combined with HTS-relevant reading rates, offers significant advantages for a broad variety of HTS workflows, including phenotype screening, biochemical and cell-based mechanistic assays, and binding assays. In addition, by accelerating HTE synthetic chemistry for streamlining molecular libraries, adding TIMS to MS-based read-outs will help ensure a constant stream of new, well-characterized drug candidates, and so improve the ability to tackle ongoing health challenges.
References
1 M.S. Attene-Ramos and M. Xia, in Encyclopedia of Toxicology (3rd edition), Elsevier, 2014.
2 R.L. Lundblad, Drug Design, in Encyclopedia of Cell Biology (Volume 1), Elsevier, 2016.
3 F. Pu, N.L. Elsen and J.D. Williams, Emerging chromatography-free high-throughput mass spectrometry technologies for generating hits and leads, ACS Medicinal Chemistry Letters, 2020, 11: 2108–2113, https://doi.org/10.1021/acsmedchemlett.0c00314.
4 D.G. McLaren, V. Shah, T. Wisniewski, L. Ghislain, C. Liu, H. Zhang and S.A. Saldanha, High-throughput mass spectrometry for hit identification: Current landscape and future perspectives, SLAS Discovery, 2021, 26: 168–191, https://doi.org/10.1177/2472555220980696.
5 M.W. Duncan, D. Nedelkov, R. Walsh and S.J. Hattan, Applications of MALDI mass spectrometry in clinical chemistry, Clinical Chemistry, 2016, 62: 134–143, https://doi.org/10.1373/clinchem.2015.239491.
6 E. Torres-Sangiao, C. Leal Rodriguez and C. García-Riestra, Application and perspectives of MALDI–TOF mass spectrometry in clinical microbiology laboratories, Microorganisms, 2021, 9: 1539, https://doi.org/10.3390/microorganisms9071539.
7 S. Castellino, M.R. Groseclose and D. Wagner, MALDI imaging mass spectrometry: Bridging biology and chemistry in drug development, Bioanalysis, 2011, 3: 2427–2441, https://doi.org/10.4155/bio.11.232.
8 M.S. Ritorto, R. Ewan, A.B. Perez-Oliva, A. Knebel, S.J. Buhrlage, M. Wightman, S.M. Kelly, N.T. Wood, S. Virdee, N.S. Gray, N.A. Morrice, D.R. Alessi and M. Trost, Screening of DUB activity and specificity by MALDI-TOF mass spectrometry, Nature Communications, 2014, 5: 4763, https://doi.org/10.1038/ncomms5763.
9 C. Haslam, J. Hellicar, A. Dunn, A. Fuetterer, N. Hardy, P. Marshall, R. Paape, M. Pemberton, A. Resemannand and M. Leveridge, The evolution of MALDI-TOF mass spectrometry toward ultra-high-throughput screening: 1536-well format and beyond, Journal of Biomolecular Screening, 2016, 21: 176–186, https://doi.org/10.1177/1087057115608605.
10 M. Winter, R. Ries, C. Kleiner, D. Bischoff, A.H. Luippold, T. Bretschneider and F.H. Büttner, Automated MALDI target preparation concept: Providing ultra-high-throughput mass spectrometry-based screening for drug discovery, SLAS Technology, 2019, 24: 209–221, https://doi.org/10.1177/2472630318791981.
11 R.P. Simon, M. Winter, C. Kleiner, R. Ries, G. Schnapp, A. Heimann, J. Li, L. Zuvela-Jelaska, T. Bretschneider, A.H. Luippold, W. Reindl, D. Bischoff and F.H. Büttner, MALDI-TOF mass spectrometry-based high-throughput screening for inhibitors of the cytosolic DNA sensor cGAS, SLAS Discovery, 2020, 25: 372–383, https://doi.org/10.1177/2472555219880185.
12 D. Weigt, C.A. Parrish, J.A. Krueger, C.A. Oleykowski, A.R. Rendina and C. Hopf, Mechanistic MALDI-TOF cell-based assay for the discovery of potent and specific fatty acid synthase inhibitors, Cell Chemical Biology, 2019, 26: 1–10, https://doi.org/10.1016/j.chembiol.2019.06.004.
13 R.E. Heap, A. Segarra-Fas, A.P. Blain, G.M. Findlay and M. Trost, Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: Development of a reproducible and robust sample preparation workflow, Analyst, 2019, 144: 6371–6381, https://doi.org/10.1039/c9an00771g.
14 R.P. Simon, M. Winter, C. Kleiner, L. Wehrle, M. Karnath, R. Ries, M. Zeeb, G. Schnapp, D. Fiegen, T.T. Häbe, F. Runge, T. Bretschneider, A.H. Luippold, W. Reindl, D. Bischoff, W. Reindl and F.H. Büttner, MALDI-TOF-based affinity selection mass spectrometry for automated screening of protein–ligand interactions at high throughput, SLAS Discovery, 2021, 26: 44–57, https://doi.org/10.1177/2472555220959266.
15 Bruker launches new timsTOF-based MALDI PharmaPulse® solution for label-free HTS in drug discovery [News release], Bruker Daltonics, 2022.
16 J.N. Dodds and E.S. Baker, Ion mobility spectrometry: Fundamental concepts, instrumentation, applications, and the road ahead, Journal of the American Society of Mass Spectrometry, 2019, 30: 2185–2195, https://doi.org/10.1007/s13361-019-02288-2.
17 Trapped Ion Mobility Spectrometry (TIMS), Bruker Daltonics, https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof.html [accessed 8 April 2022].
18 S.M. Mennen et al., The evolution of high-throughput experimentation in pharmaceutical development and perspectives on the future, Organic Process Research & Development, 2019, 23: 1213–1242, https://doi.org/10.1021/acs.oprd.9b00140.
19 A. Asperger, M. Hamester and M. Greig, High-throughput experimentation reaction monitoring and analysis of chemistry synthesis products with ultrahigh-throughput 4D timsTOF fleX MALDI-2 technology [Bruker Application Note LCMS-182], Bruker Daltonics, 2021.
20 S. Lin, S. Dikler, W.D. Blincoe, R.D. Ferguson, R.P. Sheridan, Z. Peng, D.V. Conway, K. Zawatzky, H. Wang, T. Cernak, I.W. Davies, D.A. DiRocco, H. Sheng, C.J. Welch and S.D. Dreher, Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS, Science, 2018, 361: eaar6236, https://doi.org/10.1126/science.aar6236.
21 Jens Soltwisch, Bram Heijs, Annika Koch, Simeon Vens-Cappell, Jens Höhndorf, and Klaus Dreisewerd, MALDI-2 on a Trapped Ion Mobility Quadrupole Time-of-Flight Instrument for Rapid Mass Spectrometry Imaging and Ion Mobility Separation of Complex Lipid Profiles, Analytical Chemistry 2020 92 (13), 8697-8703, DOI: 10.1021/acs.analchem.0c01747
22 timsTOF fleX MALDI-2, Bruker Daltonics, https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof/timstof-flex-maldi-2.html [accessed 8 April 2022].
